1. Field of the Invention
This invention relates to improved vaccine adjuvant compositions, improved processes for preparing said adjuvant compositions, and methods of using the improved compositions.
2. Related Disclosures
Adjuvants are useful for improving the immune response obtained with any particular antigen in a vaccine. Although some antigens are administered in vaccines without an adjuvant, there are many antigens that lack sufficient immunogenicity to stimulate an useful immune response in the absence of an effective adjuvant. Adjuvants also improve the immune response obtained from "self-sufficient" antigens, in that the immune response obtained may be increased or the amount of antigen administered may be reduced.
The standard adjuvant for use in laboratory animals is Freund's adjuvant. Freund's complete adjuvant (FCA) is an emulsion containing mineral oil and killed mycobacteria in saline. Freund's incomplete adjuvant (FIA) omits the mycobacteria. Both FIA and FCA induce exceptional humoral (antibody) immunity, and FCA additionally induces high levels of cell-mediated immunity. However, neither FIA nor FCA are acceptable for use outside the laboratory due to the adjuvants' side effects. Mineral oil is known to cause abscesses and granulomas, while Mycobacterium tuberculosis is the agent responsible for tuberculosis.
A number of naturally occurring compounds such as the lipid-A portion of gram negative bacteria endotoxin and trehalose dimycolate of mycobacteria have been tried as substitutes for FCA and FIA. Also, the phosholipid lysolecithin has been shown to have adjuvant activity (B. Arnold et al., Eur. J. Immunol., 9:363-366 (1979)). In addition, several synthetic surfactants, for example, dimethyldioctadecyl ammonium bromide (DDA) and certain linear polyoxypropylene-polyoxyethylene (POP-POE) block polymers (available commercially under the trademark Pluronic.RTM.) have been reported as having adjuvant activity (H. Snippe et al, Int. Archs. Allergy Appl. Immun., 65, 390-398 (1981)). R. Hunter et al. have reported in J. Immunol., 127, 1244-1250 (1981) that POP-POE block polymers increase antibody formation to bovine serum albumin (BSA) in mice when used as the surfactant component of an mineral oil/water emulsion adjuvant formulation. While these natural and synthetic surfactants demonstrate-some degree of adjuvanticity, they for the most part fail to achieve the degree of immunopotentiation obtained using FCA or FIA.
Taking another approach, it has been determined that the adjuvant effect from mycobacteria is due to a muramyl-peptide in the cell wall. The smallest fragment of this molecule that retains adjuvant activity is N-acetylmuramyl-L-alanyl-D-isoglutamine, commonly known as muramyl dipeptide or "MDP" (Ellouz et al, Biochem. & Biophys. Res. Comm., Vol 59, 4, 1317 (1974)). Numerous derivatives of MDP have been prepared, and are also referred to as "MDPs." See for example Audibert et al., U.S. Pat. No. 4,158,052; Audibert et al., U.S. Pat. No. 4,220,637; Audibert et al., U.S. Pat. No. 4,323,559; Baschang et al., U.S. Pat. No. 4,323,560; Baschang et al., U.S. Pat. No. 4,409,209; Baschang et al., U.S. Pat. No. 4,423,038; Derrien et al., U.S. Pat. No. 4,185,089; Hartmann et al., U.S. Pat. No. 4,406,889; Jones et al., U.S. Pat. No. 4,082,735; Jones et al., U.S. Pat. No. 4,082,736; Le Francier et al., U.S. Pat. No. 4,427,659; Le Francier et al., U.S. Pat. No. 4,461,761; Yamamura et al., U.S. Pat. No. 4,314,998; Yamamura et al., U.S. Pat. No. 4,101,536; and Yamamura et al., U.S. Pat. No. 4,369,178, all of which are incorporated herein by reference. While these compounds are weakly effective at stimulating the immune system when administered in aqueous solution, the results generally fall short of the specific immune response obtained with FIA or FCA.
A particularly effective adjuvant composition comprising a glycopeptide, a non-toxic POP-POE block polymer, a glycol ether-based surfactant, a metabolizable oil, and buffered saline was recently described by Allison et al., U.S. Pat. Nos. 4,606,918, 4,770,874, and 4,772,466, all of which are incorporated herein by reference. The adjuvant composition described by Allison et al. is capable of inducing strong humoral and cell-mediated immune responses, equivalent or superior to the results achieved in laboratory animals using FCA. However, the composition is prone to instability and separation (e.g., creaming) upon standing. We have discovered that upon refrigeration it loses its ability to potentiate the primary response to antigens. Also, it has been found difficult to prepare a stable, homogenous emulsion with retention of full adjuvant activity on a commercial scale.
We have now discovered that an immunopotentiating glycopeptide can be formulated with a non-toxic N,N,N',N'-tetra(polyoxypropylene-polyoxyethylene)-1,2-diaminoethane block polymer ("tetra-polyol"), resulting in an adjuvant composition that overcomes certain problems of the prior art, for example, toxicity and failure to stimulate cell-mediated immunity. This new adjuvant composition has activity equal or greater than the activity of FCA and Allison's composition. The composition of the present invention is easily manufactured with full retention of activity, and displays greater pH stability than Allison's composition. Because the tetra-polyol is non-toxic, this adjuvant composition may be safely used as a vehicle for enhancing the immunogenicity of antigens administered to birds and mammals.
We have also invented a particularly advantageous method for preparing an emulsion of an adjuvant composition, using either POP-POE block polymers or tetra-polyols, which maintains the composition's efficacy, enhances its physical stability, and reduces its sensitivity to refrigeration. Remarkably, such an emulsion may even be frozen and still retain efficacy.